Recent studies have shown a beneficial effect of cardiosphere-derived cell (CDC) therapy on regeneration of injured myocardium following myocardial infarction (MI). While paracrine signaling by CDC secreted exosomes (CDC-exo) is hypothesized to be the principal mediator of improved function, the contribution of exosomes secreted from grafted cells and their mechanisms of action have not been established. Our central hypothesis is that exosomes released by engrafted CDCs directly contribute to cardioprotection. We have previously shown that human CDC-derived exosomes have a primarily anti-apoptotic effect on cardiac myocytes and antiproliferative effect on cardiac fibroblasts. Furthermore, we found that macrophages primed with CDC-exo are polarized towards an anti-inflammatory phenotype with high expression of Arg1. Together, our studies support a role for exosome secretion as a paracrine mechanism of stem cell-mediated cardiac repair in vivo. Using a novel genetic lentiviral knockdown strategy to inhibit neutral sphingomyelinase 2 (nSMase2), a crucial enzyme in exosome secretion, we will test constitutive inhibition of CDC exosome secretion in vivo, enabling us to achieve our long term goal of determining the functional relevance and mechanism of cardiosphere-derived cell exosomes on the restoration of cardiac function. Success in achieving the following specific aims is of high impact as there are few pharmacological targets capable of influencing cardiac regeneration. To achieve these ends, we shall address the following specific aims: Specific Aim 1: Determine the contribution of CDC-exo vs. CDC-secretome on infarct size in a mouse model of acute MI. Specific Aim 2: Define the role of CDC-exo on macrophage polarization and cardioprotection. Specific Aim 3: Determine the role of exosomal miRNA on CDC-exo mediated cardiac repair. In summary, the goals of this work are to define the role of physiologically secreted CDC- exo on cardiac function post MI, elucidate the contribution of CDC-exo-modulated macrophage polarization on cardioprotection, and define the functional role of exosomal miRNA in CDC-exo mediated effects. In doing so, we aim to identify novel therapeutic approaches to stimulate cardiac muscle regeneration that do not require the administration of stem cell preparations and can potentially enhance the care of patients with both heart failure and prior myocardial infarction.